Method for transferring material in a cell system

ABSTRACT

Described is a method for method for transferring material through the membrane of at least one cell, wherein the transfer is carried out in the presence of trehalose. This method is applicable in particular in the field of genetic engineering and biotechnology.

The present invention relates to a method for transferring materialthrough the membrane of at least one cell, as well as to the applicationof this method in genetic engineering, biotechnology and hybridomatechnology.

Over the past few years, methods for transferring biological materialsthrough the membrane of a cell have increasingly gained in importance.In these methods, membrane-impermeable molecules are transferred throughpores that have formed in the membrane as a result of extraneous forces.These methods are associated with a decisive advantage in that there isno need for the use of any vehicles.

Reversible membrane permeabilization through an electrical field, oralternatively, electroporation, has for some time been an establishedmethod of taking up free DNA, for example in eukaryotes. In thisprocess, eukaryotes, in the presence of DNA, are exposed to ahigh-strength electrical field. However, only very little is known aboutthe mechanism of DNA uptake during electroporation. It is generallyassumed that due to the electric shock, pores form temporarily in thecell membrane, and the DNA, after contact with the lipid bi-layer of thecell membrane, is taken up into the cell.

While in electroporation, material is imported to the cell from theexterior, electrofusion differs in that at least two cells fuse.Electrofusion takes place by means of electrical pulses in two stages.In the first step, the cells to be fused are subjected to an alternatingelectrical field in which they are mutually attracted to each other as aresult of dielectrophoresis. Conductivity of the medium should be as lowas possible. In the second step, electrofusion is triggered by veryshort electrical direct-current pulses. This leads to interaction ofmembrane parts which leads to fusion. With this method it is possible,for example, to fuse protoplasts. It is also possible to produce hybridsof animal cells, such as hybridoma cells, as well as yeasts.

It has thus been known for some time to cause transfer of material intoa cell in that the cells are subjected to irradiation treatment for thepurpose of permeabilization. In this process, the cells are for examplesubjected to laser irradiation, after which the material can thendiffuse through the cell membrane.

Methods have thus already been used in which, for the purpose ofpermeabilization, the membranes of cells are treated with chemicalsubstances. Such substances include pore-forming and/ordiffusion-promoting peptide antibiotics and depsipeptide antibiotics,such as valinomycin, and detergents, such as sodium dodecyl sulfate.

The methods described above are based on the following fundamentalprinciple: local apertures in the cell membrane result fromenergy-intensive electrical, electromagnetic, or mechanical forces suchas current pulses, irradiation, ultrasound and pressure, and chemicaltreatment. Submicroscopic holes or pores occur to form. Such treatmentmakes possible introducing the biological material from the exterior,or, between cells, if two cells are to be fused. After diminishedintensity of these forces acting from the exterior, the pores in themembrane close up, and the material remains in the cell.

However, it has been shown that in this method, the fraction ofsurviving cells, i.e. intact cells which have been reversiblypermeabilized, is often exceeded by the fraction of dead cells. This isdue to the permeabilized cells being unable to completely re-close thepores after a reduction in the intensity of the external forces. Thisthen leads to the loss of important cell functions so that the cell isno longer able to maintain its metabolism, a situation which finallyleads to the death of the cell. For this reason, it was quite normal inthe method according to the state of the art, to have to accept afraction of dead cells, a factor which had a significant negative effecton the efficiency of this method.

It is thus the object of the present invention to provide a method fortransferring material through the membranes of cells, by which a highdegree of reversibly permeabilized, so-called surviving cells isachieved, and thus the fraction of dead cells is drastically reduced. Anincrease in the fraction of surviving cells should also be realized inthe event of working with more stringent reaction conditions, forexample increased field strengths.

This object is solved by the method according to claim 1. The sub-claimsrelate to preferred embodiments of the method according to theinvention.

Furthermore, the claims describe particular applications of the methodaccording to the invention.

The present invention relates to a method for transferring materialthrough the membrane of at least one cell, in an aqueous medium which ischaracterized in that the transfer is carried out in the presence oftrehalose.

The method according to the invention is explained in more detail withreference to the accompanying figures. The following are shown:

FIG. 1—a graphic representation showing the propidium iodide uptake andthe survival rate (% reversibly permeabilized cells) depending on thefield strength in the hypo-osmolar medium;

FIG. 2—a graphic representation showing the “pulse efficiency” as ameasure of the yield, depending on the field strength in thehypo-osmolar medium;

FIG. 3—a graphic representation showing the propidium iodide uptake andthe survival rate (% reversibly permeabilized cells) depending on thefield strength in the iso-osmolar medium; and

FIG. 4—a graphic representation showing the “pulse efficiency” as ameasure of the yield, depending on the field strength in the iso-osmolarmedium.

According to the invention it has been shown that the survival rate ofreversibly permeabilized cells is drastically increased if trehalose isadded to the aqueous working medium. Presumably, the trehalose has amembrane-stabilizing and membrane-healing effect after the closure ofthe pores following introduction of the biological material.Consequently, the permeabilized cells survive and there is thus nodanger of cell functions dying due to the loss of cell fluids and cellorganelles.

The use of trehalose in methods of the type described here has not beenknown up to now. The literature merely indicates that trehalose makes acontribution to the cryoconservation of mammalian cells (NatureBiotechnology, vol. 18, February 2000) and to sustaining intact humancells without the presence of water (Nature Biotechnology, vol. 18,February 2000).

In principle, any trehalose which is dissolved in the respective workingbuffers is suitable. Trehalose is a disaccharide which occurs naturally;in one case it is also made synthetically. α,α-trehalose is thebest-known trehalose and the one that most frequently occurs naturally.α,β-trehalose also occurs naturally; it has been found to be present inhoney. β,β-trehalose is only available synthetically. Preferably,α,α-trehalose is added to the working buffer for transferring material.

The method according to the invention is preferably suitable for thetransfer of material in which at least one cell is involved which isreversibly permeabilized, or in which at least two cells which adhere toeach other are involved which are permeabilized and which practicallyexchange material between each other. In this case it is possible thatat least two cells fuse. There is also a further variant where severalcells fuse, quasi by forming a string of pearls.

Normally, transfer of material into the cell is through a local apertureor apertures in the membrane of the cell or the cells. This is calledpermeabilization of the membrane. Presumably material is transferredthrough part of the apertures while the other part of the aperturesremains without material passage. Having taken up the material, theseapertures too, have to close. It is especially in the context of thisprocess that trehalose has proven to be particularly advantageous as amembrane-healing additive.

As a rule, permeabilization of the membrane can take place by applyingan electrical field, by irradiation, or by chemical treatment. Theactual method selected essentially depends on the type of cells to betransformed and the type of material to be transferred.

Methods including electrotransfection, electroporation, orelectrofusion, either in macroscopic devices or inmicrosystems/microstructures, are suitable for electricalpermeabilization. These are established methods which have beensuccessfully in use for some years in gene technology. In particular inthe case of electroporation it is often necessary to work with highfield strengths. However, this has traditionally been associated with aproblem in that the cells subsequently died, i.e. that they had beenirreversibly permeabilized. This led to considerable reductions inyield. However, if, according to the invention, trehalose is added tothe electroporation buffer, it is possible to obtain reversiblypermeabilized living cells, even at high field strengths. This factorrenders the method according to the invention far more economical.

Thus, permeabilization by way of UV irradiation or laser irradiation isalso possible. This type of irradiation is advantageous when due to thecells used and due to the material to be transferred, electricaltreatment is not indicated.

Thus, chemical treatment for permeabilization is recommended only whenit is not advisable to carry out transfer in the electrical field ortransfer by way of irradiation. If chemical treatment is to be used,then, for permeabilization, the cells are to be treated withantibiotics, detergents, etc.

With the method according to the invention, any suitable biologicalmaterials can be transferred, including: xenomolecules, DNA and RNA,plasmids, chromosomes, parts of chromosomes as well as artificialchromosomes, proteins and glycoproteins, cells, parts of cells, and cellorganelles, or low-molecular foreign matters.

The biological material can be trehalose or a trehalose/saccharosemixture. In this way it is possible to introduce trehalose or thetrehalose/saccharose mixture as an intracellular cryoprotectant orprotectant against desiccation.

As far as the cells used during transfer are concerned, the methodaccording to the invention is not subject to any limitations. It ispossible to use natural cells or membrane-enveloped vesicles for thetransfer of material. Natural or artificial vesicles, liposomes andmicelles are examples of membrane-enveloped vesicles.

In the case of natural cells, according to the invention it is possibleto permeabilize and transform prokaryotic and eukaryotic cells.Bacteria, blue algae and archae-bacteria are examples of prokaryoticcells. Eukaryotic cells can have their origins in protozoa, plants(including algae), fungi (including yeasts), animals or humans.

For electroporation and electrofusion, it is possible to work in theiso-osmolar medium or in the hypo-osmolar medium. In animal cells, thehypo-osmolar medium has an osmolarity of 75 to 250 mOsm and is thusnonphysiological. The osmolarity of an iso-osmolar medium is approx. 300mOsm, with the medium corresponding to the physiological environment. Incontrast, in the case of plant cells, the osmolarity of an iso-osmolarmedium is approx. 500 mOsm. The hypo-osmolar medium ranges from approx.400 to 450 mOsm.

It has been found that the protective effect of trehalose becomesevident in particular in the hypo-osmolar medium.

It has been shown-that the concentration of trehalose in an aqueousmedium, for example in the case of electrical treatment, should bewithin the range of 1 to 200 mM. It has been found that trehaloseincreases the fraction of surviving cells after pulse application, withoptimal yield occurring at approx. 30 to 50 mM which is not increased byfurther increasing the concentration level. Therefore, a concentrationrange of approximately 30 to 50 mM is preferred for pulse application.

It has been shown that a further embodiment of the method according tothe invention brings about increased yield of reversibly permeabilizedcells even if a mixture of trehalose and saccharose is added to theworking buffer. The ratio of trehalose to saccharose ranges from 1:2 to1:10. The concentration in the mixture ranges from 200 to 300 mM.

The present method is eminently suitable for introducing biologicalmaterial into a cell, or for transfer between at least two cells. Themethod can therefore be applied in practically all areas ofbiotechnology, genetic engineering and microsystem technology. Inparticular, it is especially suited to hybridoma technology, for examplewhere elecrofusion is used. Furthermore, plant protoplasts can easily befused with the method according to the invention.

As a result of the presence of trehalose in the working medium, themethod according to the invention has many advantages. These are due tothe fact that trehalose has a protective effect on the cells to betreated. Trehalose stabilizes the cell membrane and causes fast healingof the pores, for example following the uptake of foreign material andweakening of the external forces. In particular in the case ofelectroporation it has been found that the protective effect oftrehalose is very pronounced at high field strengths, while at the sametime hardly being affected by the pulse duration. It has been shown thatthe protective effect of trehalose is stronger in the hypotonic pulsemedium than it is in the isotonic pulse medium. The effect of trehaloseis somewhat more pronounced in a poorly conductive medium than it is ina stronger conducting medium. These advantageous protective propertiesof trehalose are in particular highly beneficial if work is carried outat stringent pulse conditions, such as poor conductivity, hypotonicstress, or high field strength.

Below, the method according to the invention is explained in more detailby means of examples.

EXAMPLES Example 1

Electroporation of Cells with and without Trehalose in a Hypo-osmolarWorking Medium.

A phosphate buffer with 1.15 mM K₂HPO₄/KH₂PO₄ buffer, pH 7.2, was usedas a pulse medium. KCl at a concentration of 10 mM (α=1.5−1.6 mS/cm) wasadded as a conducting salt. After this, trehalose at the respectiveconcentration was added to the pulse medium. Osmolarity was adjusted to100 mOsm by the addition of inositol, so as to obtain a hypo-osmolarsolution.

Jurkat cells that are cells of a human T-lymphocytes line were used. 40μg/ml propidium iodide, which is a membrane-impermeable DNA dye, wasadded as the material to be transferred.

Pulse application took place after 10 minutes of incubation prior topulse application, in the pulse medium at room temperature (celldensity: 2-3×10⁶ cells/ml). Pulse duration was 20 μs.

Electroporation took place in an Eppendorf multiporator. After pulseapplication, the pores were left to reseal for 10 min at roomtemperature.

Electropermeabilisation took place at the following trehaloseconcentrations: 0 mM and 40 mM (α,α-trehalose).

Pulsing takes place at 4° C. or at room temperature. This can be singlepulsing; however, multiple pulsing involving up to 3 pulses can at timesbe advantageous.

The results are shown in FIGS. 1 and 2.

FIG. 1 diagrammatically shows the uptake of propidium iodide or thesurvival rate of the electropermeabilized cells depending on the fieldstrength. Poration without trehalose is shown by squares. Preparationwith 40 mM Trehalose is shown by triangles. Outline symbols designatethe survival rate (percentage of reversibly-permeabilized cells) whilesolid symbols designate the propidium iodide uptake into the cell.

As shown in FIG. 1, in a pulse medium without trehalose, cells sustainirreversible damage at field strengths from 1.5 kV/cm onwards, anddie-off as a result of loss of cell functions (outline squares). Incontrast, in a pulse medium in 40 mM trehalose, there is only arelatively small percentage of dead cells (outline triangles), even atvery high field strengths up to 2.5 kV/cm. This is an indication thatthe cells were reversibly permeabilized and have thus remained viable.Propidium iodide uptake is only slightly influenced by trehalose.

FIG. 2 depicts an investigation of the pulse efficiency which representsthe product of propidium iodide uptake and survival rate, as a measureof the yield obtained from electroporation, depending on the fieldstrength. It is evident that with 40 mM trehalose in the pulse medium(triangles), these values rise rapidly as the field strength increases,and are significantly above the values of a control sample (squares)which was not treated with trehalose. This significant increase in yieldis also due to a greatly improved survival rate in the presence of 40 mMtrehalose.

Example 2

Electroporation of Cells with and without Trehalose in an Iso-osmolarWorking Medium.

Essentially, the same experimental conditions as in Example 1 applied,except that the osmolarity of the pulse medium was adjusted toiso-osmolar conditions by adding inositol to 290 mOsm.

Again, α,α-trehalose was added in the following concentrations: 0 mM and40 mM.

The results are shown in FIGS. 3 and 4.

FIG. 3 shows the dependence of the survival rate and the propidiumiodide uptake in relation to the field strength. Outline symbolsdesignate the survival rate, while solid symbols designate the propidiumiodide uptake.

In the iso-molar pulse medium, the survival rate in the case ofuntreated cells (0 mM, outline squares) is many times less than in thecase of cells treated with 40 mM trehalose (outline triangles), aboveall at high field strength. In contrast, propidium iodide uptake variesless markedly. It is important to note that from a field strength of 3kV/cm onwards, there is a drastic die-off of cells if there is notrehalose in the pulse medium.

FIG. 4 shows the pulse efficiency dependent on the field strength forthe present pulse media (0 to 40 mM trehalose). As the field strengthincreases, a gradual increase in the pulse efficiency is evident in thepulse medium containing trehalose (triangles), while without trehalose(squares) in the pulse medium at high field strength, a drasticreduction in yield is evident. Here again, trehalose displays itsprotective effect in stringent pulse conditions.

1. A method for transferring a material selected from the groupconsisting of xenomolecules, DNA, RNA, plasmids, parts of chromosomes,proteins, and glycoproteins, through a membrane of at least one cell inan aqueous medium, said method comprising: permeabilizing the membraneby applying an electric field to create a permeabilized membrane; andallowing a transfer of the material through the permeabilized membranein the aqueous medium, and wherein the aqueous medium comprises aconcentration of 1-200 mM trehalose and is: (a) an iso-osmolar mediumand the electric field has a strength of 3 to 4 kV/cm, or (b) ahypo-osmolar medium and the electric field has a strength of 1.5 to 2.5kV/cm.
 2. The method according to claim 1, wherein the transfer of thematerial comprises moving the material from an exterior of the at leastone cell into an interior of the at least one cell, or wherein thematerial is transferred between at least two cells.
 3. The methodaccording to claim 1, wherein the material is obtained from a biologicalsource.
 4. The method according to claim 1, wherein the at least onecell is a living cell.
 5. The method according to claim 4, wherein theat least one cell is at least one of a prokaryotic living cell and aneukaryotic living cell.
 6. The method according to claim 5, wherein theeukaryotic living cell is of human, animal or plant origin.
 7. Themethod according to claim 1, wherein the concentration of trehalose is30 to 50 mM.
 8. The method according to claim 1, wherein the material isobtained from a biological source, and the method is employed in a fieldselected from the group consisting of genetic engineering,biotechnology, hybridoma technology and microsystem technology.
 9. Themethod according to claim 1, wherein the trehalose does not contributeto cryoconservation of the cell.